Effects of membrane potential and cytosolic free Ca 2 + concentrations ([Ca 2 + ] i ) on acetycholine (ACh)-induced inositol phosphate production were investigated in insulin secreting βTC3 cells. ACh (10 μM) caused a rapid inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) production and increase in [Ca 2 + ] i reaching a maximum within 5 s. The rise in Ins(1,4,5)P 3 production was reduced by 79 ± 5% when [Ca 2 + ] i was kept low in cells loaded with the Ca 2 + chelator BAPTA. The ACh-evoked Ins(1,4,5)P 3 production also depended on the membrane potential as it was reduced by 31 ± 6% in cells hyperpolarized by diazoxide, an opener of ATP-sensitive K + channels. The Ca 2 + ionophore ionomycin caused a rapid increase in [Ca 2 + ] i and in the cellular Ins(1,4,5)P 3 content. We conclude that stimulation-induced changes in membrane potential and [Ca 2 + ] i play an important role in controlling Ins(1,4,5)P 3 production in insulin-secreting βTC3 cells.